Signaling inputs that maintain the heterogeneity of colorectal cancer cells Abstract Butyrate, a fermentation product of dietary fiber in the colon, inhibits the activity of histone deacetylases and induces cell growth arrest, differentiation, and apoptosis in colorectal cancer (CRC) cells in vitro. Based upon these activities, butyrate is a likely mediator of the protective role of dietary fiber against colon cancer. We have established that the apoptotic levels in CRC cells exposed to butyrate depend upon the levels to which the agent hyper-activates Wnt signaling, a pathway that is abnormally activated in most CRCs. However, within each CRC cell population, only a fraction of cells acquires high levels of Wnt signaling, and the apoptotic outcome is limited to this fraction. Similar Wnt signaling heterogeneity of CRCs in vivo may contribute to their resistance to the apoptotic effects of butyrate and synthetic histone deacetylase inhibitors designed as anti-cancer agents. Based upon preliminary studies, we hypothesize that exposure to butyrate induces a process of cell-fate assignment similar to this of lateral inhibition. However, due to modified signaling inputs, this process does not result in the terminal differentiation of all cells: only cells with hyper-induced Wnt signaling undergo apoptosis;whereas, cells with moderate levels of Wnt signaling acquire more invasive phenotype supported by noncanonical TGFbeta/Activin, Notch, and Wnt signaling pathways. The aims of the project are: (1) to investigate the role for noncanonical TGFbeta/Activin signaling in CRC cells with differential sensitivity to the effects of butyrate, (2) to determine how components of Notch pathway maintain the heterogeneity in butyrate-treated CRC cell populations in terms of Wnt signaling levels, and (3) to test a bimodal approach that targets with apoptosis both low- and high-Wnt signaling CRC cells. For our analyses on the transcriptional activity and apoptotic role of the three signaling pathways we will utilize a panel of human CRC cell lines with differential sensitivity to the apoptotic effect of butyrate, as well as developed by us butyrate-resistant cells. The efficacy of a bimodal approach that targets with apoptosis both cells with low and high Wnt signaling will be tested in a 3-dimensional cell culture system. Understanding the interactions between noncanonical TGFbeta/Activin, Notch and Wnt signaling pathways may suggest approaches to overcome the CRC cell heterogeneity by targeting cells with both high and low sensitivity to the apoptotic effect of butyrate. Such strategies will be valid for an increasing cohort of synthetic histone deacetylase inhibitors, compounds that mimic the effects of butyrate on Wnt signaling and apoptosis in CRC cells and are currently in clinical trials as anti-cancer therapeutic agents. PUBLIC HEALTH RELEVANCE: Colorectal cancer cell populations in vitro and in vivo are heterogeneous in terms of Wnt signaling levels. This heterogeneity renders some of the cells more resistant to the programmed cell death induced by butyrate, a fermentation product of dietary fiber in the colon, and other anti-cancer agents, which mimic the effects of butyrate. We will analyze the mechanisms via which colorectal cancer cell heterogeneity arises, and test a novel treatment approach to overcome it.